Protective effect of galangin in Concanavalin A-induced hepatitis in mice

Abstract

Galangin is an active pharmacological ingredient from propolis and Alpinia officinarum Hance, and has been reported to have anti-inflammatory and antioxidative properties. The present study aims to reveal the effect of galangin on Concanavalin A (ConA)-induced hepatitis (CIH), a well-established animal model of immune-mediated liver injury, and to clarify the related mechanism. C57BL/6 mice were pretreated with galangin followed by ConA challenge. Results indicated that galangin inhibited ConA-induced liver damage. Mice pretreated with galangin showed more reduction of liver damage when compared with control mice pretreated with vehicle solution. In galangin-pretreated mice with induced CIH, increases in serum levels of several inflammatory cytokines, including tumor necrosis factor-α, interferon-γ, and interleukin-12 were dramatically attenuated, and chemokines and adhesion molecules like interferon inducible protein-10, macrophage inflammatory protein-1α, and inter-cellular adhesion molecule-1 messenger RNA expressions in liver were decreased. Moreover, CIH mice pretreated with galangin showed less leukocyte infiltration and T-cell activation in the liver. Further, the mechanism of the anti-inflammatory effects of galangin may be attributed to its modulation of crucial inflammatory signaling pathways, including nuclear factor kappa B and interferon-gamma/signal transducer and activator of transcription 1. Collectively, these findings suggest the preventive and therapeutic potential of galangin in immune-mediated liver injury in vivo.

Keywords: Concanavalin A-induced hepatitis; STAT1; galangin; nuclear factor kappa B.

Figure 1

Effect of galangin on serum aminotransferases. Notes: (A) Molecular structure of galangin. (B) C57BL/6 mice, n=5 per group, received vehicle or galangin (25 or 50 mg/kg) by intraperitoneal injection. Four hours later, galangin injection was repeated. Control mice received equivalent vehicle solution accordingly. After the second injection of galangin or vehicle, all mice were injected with a single dose (15 mg/kg) of Concanavalin A via the tail vein. Mice were euthanized at the indicated time points and serum levels of ALT and AST were measured. The results are shown as the mean ± standard deviation. Data are representative of three experiments. *P<0.05, **P<0.01, ***P<0.001. Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase.

Figure 2

Liver H&E and TUNEL staining. Notes: Eight hours after Concanavalin A injection, liver tissues were obtained and sectioned, then stained by H&E or TUNEL. H&E staining for (A) vehicle-pretreated mice and (B) galangin-pretreated mice. TUNEL staining for (C) vehicle-pretreated mice and (D) galangin-pretreated mice. Ten sections were observed in each animal. Results are representative of five experiments. (H&E, magnification ×100; TUNEL, magnification ×200). Abbreviations: H&E, hematoxylin-eosin; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling.

Figure 3

Influence of galangin on serum cytokines. Notes: C57BL/6 mice received vehicle or galangin pretreatment and ConA injection. (A) Two hours after injection of ConA, serum was obtained and TNF-α was measured by ELISA. Eight hours after ConA injection, serum IFN-γ (B) and IL-12 (C) were measured by ELISA. Data are shown as the mean ± standard deviation. The results are representative of three experiments. **P<0.01, ***P<0.001. Abbreviations: ConA, Concanavalin A; ELISA, enzyme-linked immunosorbent assay; IFN-γ, interferon gamma; IL-12, interleukin-12; TNF-α, tumor necrosis factor alpha.

Figure 4

Effects of galangin on chemokine and adhesion molecule messenger (m)RNA expression. Notes: Eight hours after injection of Concanavalin A, liver RNA from mice was obtained and mRNA expression levels of (A) IP-10, (B) MIP-1α, and (C) ICAM-1 were quantitatively analyzed by real-time polymerase chain reaction. Results are representative of three experiments. *P<0.05, **P<0.01. Abbreviations: IP-10, IFN-inducible protein-10; MIP-1α, macrophage inflammatory protein-1 alpha; ICAM-1, intercellular adhesion molecule-1.

Figure 5

Influence of galangin on recruitment and activation of inflammatory cells. Notes: (A) IHLs were isolated from vehicle-pretreated CIH mice and galangin-pretreated CIH mice (2 hours after injection of Concanavalin A). The total number of IHLs from each mouse was counted using a cell counting plate. IHLs were stained with CD3e, CD11b, Gr-1, and NK1.1. The number of each cell subset was calculated by multiplying the total number of IHLs by the frequency of the subset. (B) IHLs were stained with CD3e and CD69, and the percentage of activated T-cells was analyzed. Data are shown as the mean ± standard deviation. The results are representative of three experiments. *P<0.05, **P<0.01. Abbreviations: CIH, Concanavalin A-induced hepatitis; FITC, fluorescein isothiocyanate; APC, allophycocyanin; PE, R-phycoerythrin; SCC-H, side scatter-height; FSC-H, forward scatter-height; IHLs, intrahepatic leukocytes.

Figure 6

Effect of galangin on NF-κB and STAT1 activation. Notes: Mice received vehicle or galangin pretreatment and ConA injection. (A) Two hours after ConA injection, IκBα, pIκBα, STAT1, and pSTAT1 proteins in liver tissue were analyzed by Western blotting. (B) Band intensities of pIκBα and pSTAT1 were quantified by ImageProPlus 5.0 software. Densitometry confirms the decrease in IκBα and STAT1 phosphorylation in response to galangin. The results are representative of three experiments. ***P<0.001. Abbreviations: ConA, Concanavalin A; NF-κB, nuclear factor kappa B; STAT1, signal transducer and activator of transcription 1; p, phosphorylated.